One of the main applications of light sheet microscopy lies in imaging midsized specimens, for example organisms, with dimensions of several 100 μm up to a few millimeters. As a rule, these specimens are embedded in agarose and arranged in a glass capillary. For the purposes of examining the specimen, the glass capillary is introduced into a water-filled specimen chamber and the agarose with the specimen is pressed a little out of the capillary. The specimen is illuminated by a light sheet. The fluorescence that is excited in the specimen and that emanates from the latter is imaged onto a detector, in particular a camera, by means of a detection objective, which is perpendicular to the light sheet and consequently also perpendicular to the light sheet optical unit.
In accordance with the prior art, a layout of a microscope 1 for light sheet microscopy (SPIM layout; single plane illumination microscopy) comprises an illumination objective 2 with a first optical axis A1 and a detection objective 3 with a second optical axis A2 (also referred to as SPIM objectives below) which are each directed onto the specimen plane 4 from above at an angle of 45° in relation to a specimen plane 4 and at right angles in relation to one another (see FIG. 1a). A specimen 5 arranged in the specimen plane 4 is situated, for example, on the base of a specimen holder 7 embodied as a petri dish. The specimen holder 7 is filled with a liquid 8, for example water, and the two SPIM objectives 2, 3 are immersed in the liquid 8 during the application of the light sheet microscopy (not shown). The specimen plane 4 extends in an XY plane spanned by the X-axis X and the Y-axis Y of a Cartesian coordinate system. The first optical axis A1 and the second optical axis A2 extend in a plane YZ spanned by the Y-axis Y and the Z-axis Z of the Cartesian coordinate system.
This approach offers the advantage of a high resolution in the axial direction since a thin light sheet 6 may be produced by means of the illumination objective 2 and possibly further optically effective elements. Smaller specimens 5 may be examined on account of the higher resolution. Additionally, the bothersome background fluorescence is significantly reduced and the signal-to-noise ratio is improved as a result thereof.
In order to facilitate simpler specimen preparation in standard specimen containers such as e.g. multiwell plates, it is possible to maintain the 45° configuration but have the two SPIM objectives 2, 3, in an inverted arrangement, be directed into the specimen plane 4 from below through the transparent base of the specimen holder 7 (FIG. 1b). In this arrangement, it is necessary to correct the aberrations caused by the specimen holder 7 which is inclined relative to the optical axes A1 and A2 and present in the form of a cover slip by using special optical elements. The specimen 5 arranged in the specimen plane 4 is illuminated through the base of the specimen holder 7 and excited fluorescence of the specimen 5 is detected. It is possible to use specimen holders 7 such as e.g. multiwell plates, Petri dishes and/or object supports and contamination of the specimens 5, in particular in the case of high-throughput screening, may be avoided.
Further technical difficulties occur if, e.g., so-called Alvarez plates are arranged as correction elements 12 (FIG. 1B) in the beam path of the illumination objective 2 and/or of the detection objective 3 (U.S. Pat. No. 3,305,294 A). The Alvarez plates 12 are embodied in such a way that they correct aberrations that may occur, precisely in the case of a set angle between the specimen holder 7, e.g., a cover slip, and the optical axes A1, A2 of the respective objective 2, 3. Unwanted aberrations that lead to a lower imaging quality already occur in the case of a small deviation of the angle (e.g., <0.1°). Therefore, the cover slip, for example, must be aligned before the start of an experiment so that the angle deviation lies within the admissible tolerances. Moreover, it is helpful if the distance between the objective 2, 3, or a possibly present additional lens (e.g., a meniscus lens), and the cover slip is also adjustable in addition to the angle such that the specimen 5, or the region thereof to be imaged, lies in the image plane BE of the detection objective 3.
A possibility for correcting aberrations of a microscope caused by a cover slip are known from the publication by McGorty et al. (2015: Open-top selective plane illumination microscope for conventionally mounted specimens; OPTICS EXPRESS 23: 16142-16153). The inverted SPIM microscope has a water prism, by the effect of which aberrations occurring as a consequence of the oblique passage of the detection light through the cover slip are partly compensated.
A possible method for positioning a specimen holder in a beam path of a microscope is described in DE 10 2016 212 019, which has not been published to date. In the methods disclosed therein, reflected components of an illumination radiation are used to capture current actual positions and actual relative positions of the specimen holder.
In addition to the manner of positioning of the specimen holder, aberrations may also be caused by deviations of the actual thickness of the specimen holder from a nominal thickness. Commercially available cover slips, and also the bases of the Petri dishes, multiwell plates and similar specimen holders (subsumed below as specimen holders) have an allowable variance of the glass or material thickness around the nominal thickness. Thus, for example, the thickness range in the case of cover slips with the thickness #1.5 is specified as 160-190 μm, while the thickness #1 is specified as 130-160 μm. Thus, for example, if the correction element was designed for or set to a nominal thickness of 175 μm, a cover slip or base, for example a glass base, with a thickness of 160 μm leads to significant aberrations, in particular at high numerical apertures, within the scope of the oblique passage of illumination radiation, for example, through the specimen holder, which is required for the inverted configuration.
The problem here is that the thickness of the cover slip or the base of the specimen holder is not known a priori and therefore technical solutions are required to establish and set the current thickness and, optionally, the required correction parameters for the adaptive correction element.